利用 mRNA 脂质纳米颗粒对血液祖细胞中的巨核细胞进行基因工程改造。

IF 5.5 2区 医学 Q1 HEMATOLOGY Journal of Thrombosis and Haemostasis Pub Date : 2024-09-26 DOI:10.1016/j.jtha.2024.09.008
Jerry Leung, Asel Primbetova, Colton Strong, Brenna N Hay, Han Hsuan Hsu, Andrew Hagner, Leonard J Foster, Dana Devine, Pieter R Cullis, Peter W Zandstra, Christian J Kastrup
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引用次数: 0

摘要

背景:血小板是控制和处理出血的重要组成部分,通过工程改造血小板来表达治疗蛋白可扩大血小板作为细胞疗法的用途。基因工程血小板可通过改造血小板前体细胞巨核细胞(MK)来实现。目前的策略包括在体外用携带品系驱动转基因的病毒载体转染巨核细胞祖细胞,诱导产生 "体外 "改造血小板。然而,病毒的使用给临床应用带来了挑战,目前还没有利用非病毒技术对 MK 进行基因改造的方法。脂质纳米颗粒(LNP)是一种非病毒递送系统,能以简便的策略用各种核酸载荷修饰 MKs:目的:研究LNP是否能转染培养的造血干细胞/祖细胞(HSPC)衍生的MK,使其表达外源蛋白并诱导功能变化:方法:用含有mRNA的脂质纳米颗粒制剂(类似于临床批准的LNP制剂)处理从脐血衍生的HSPC分化而来的MK和MK祖细胞。转染效率通过增强型绿色荧光蛋白表达的流式细胞术进行评估。通过表达外源性凝血因子 VII(FVII)(一种具有代表性的生理相关蛋白),采用旋转血栓弹力仪评估 MK 的功能变化:结果:LNP 可使 MK 的转染效率达到 99%,并且不会影响 MK 的成熟、活力和形态。表达外源 FVII 的 MK 在 FVII 缺乏的血浆中可缩短凝血开始后的凝血时间:这种方法提供了一个易于使用的模块化平台,用于对 MK 和 MK 祖细胞进行基因修饰,并有可能扩展到生产基因修饰的培养血小板。
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Genetic engineering of megakaryocytes from blood progenitor cells using messenger RNA lipid nanoparticles.

Background: Platelets are an essential component of hemorrhage control and management, and engineering platelets to express therapeutic proteins could expand their use as a cell therapy. Genetically engineered platelets can be achieved by modifying the platelet precursor cells, megakaryocytes (MKs). Current strategies include transfecting MK progenitors ex vivo with viral vectors harboring lineage-driven transgenes and inducing the production of in vitro modified platelets. The use of viruses, however, poses challenges in clinical implementation, and no methods currently exist to genetically modify MKs with nonviral techniques. Lipid nanoparticles (LNPs) are a nonviral delivery system that could enable a facile strategy to modify MKs with a variety of nucleic acid payloads.

Objectives: To investigate whether LNPs can transfect cultured hematopoietic stem/progenitor cell-derived MKs to express exogenous proteins and induce functional changes.

Methods: MK and MK progenitors differentiated from cord blood-derived hematopoietic stem/progenitor cells were treated with LNP formulations containing messenger RNA and resembling the clinically approved LNP formulations. Transfection efficiency was assessed through flow cytometry by expression of enhanced green fluorescent protein. Functional changes to the MKs were assessed through rotational thromboelastometry by expression of exogenous coagulation factor (F)VII, a representative physiologically relevant protein.

Results: LNPs enabled transfection efficiencies of 99% in MKs and did not impair MK maturation, viability, and morphology. MKs engineered to express exogenous FVII decreased clotting time in FVII-deficient plasma following clot initiation.

Conclusion: This approach provides an easy-to-use modular platform to genetically modify MK and MK progenitors, which can be potentially extended to producing genetically modified cultured platelets.

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来源期刊
Journal of Thrombosis and Haemostasis
Journal of Thrombosis and Haemostasis 医学-外周血管病
CiteScore
24.30
自引率
3.80%
发文量
321
审稿时长
1 months
期刊介绍: The Journal of Thrombosis and Haemostasis (JTH) serves as the official journal of the International Society on Thrombosis and Haemostasis. It is dedicated to advancing science related to thrombosis, bleeding disorders, and vascular biology through the dissemination and exchange of information and ideas within the global research community. Types of Publications: The journal publishes a variety of content, including: Original research reports State-of-the-art reviews Brief reports Case reports Invited commentaries on publications in the Journal Forum articles Correspondence Announcements Scope of Contributions: Editors invite contributions from both fundamental and clinical domains. These include: Basic manuscripts on blood coagulation and fibrinolysis Studies on proteins and reactions related to thrombosis and haemostasis Research on blood platelets and their interactions with other biological systems, such as the vessel wall, blood cells, and invading organisms Clinical manuscripts covering various topics including venous thrombosis, arterial disease, hemophilia, bleeding disorders, and platelet diseases Clinical manuscripts may encompass etiology, diagnostics, prognosis, prevention, and treatment strategies.
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